Abstract Faithful replication of the genome is vital for survival. During S phase, DNA synthesis occurs at replication forks. When facing DNA damage or other impediments in chromosomes, forks are stalled creating replication stress. This stress often induces DNA double-strand breaks, which can lead to genomic instability when improperly repaired. To cope with this insult to genomic integrity, cells have evolved mechanisms to resolve stalled forks and repair double-strand breaks. Both of these mechanisms are regulated by the ATR kinase. In particular, ATR is important for the stability of fragile sites, which are specific genomic regions prone to breakage and mutagenesis when replication is perturbed. Recent evidence suggested that replication stress and double-strand breaks can be induced by R-loops, a three-stranded structure consisting of a RNA:DNA hybrid and displaced single-stranded DNA. R-loops arise from certain genes during transcription when RNA:DNA hybrids are stably formed. When occurring above normal levels, R-loops may cause transcription-associated mutagenesis, interfere with DNA replication, and lead to breakage at fragile sites. Given that R-loops may give rise to both replication stress and double-strand breaks at fragile sites, and that ATR protects fragile sites from breakage, I hypothesis that ATR is an important sensor of R-loops, and suppresses R-loop-associated genomic instability. My preliminary data suggest that ATR is activated when R-loops accumulate. Based on this finding, my Aim 1 will explore how R-loop formation triggers ATR activation. My preliminary data also show that ATR inhibition in cells with elevated R-loop levels increases DNA breaks, suggesting that ATR protects the genome from R-loop- induced DNA damage. I will investigate how ATR performs this role in Aim 2. The experiments in these two aims may reveal and elucidate the function of a key component in the DNA damage response to R-loops. Additionally, I found that R-Loop Forming Sequences are enriched in genes at early replicating fragile sites. One of these genes, IRF4, is frequently involved in oncogenic translocations in lymphomas and is predicated to form a very long R-loop. In my Aim 3, I will inducibly turn on IRF4 expression at its chromosomal locus, test if it generates R-loops, and determine if ATR protects it from R-loop-induced fragility. These experiments may reveal the importance of transcription and subsequent R-loop formation to instigating fragility at specific chromosomal loci. Given that many genes at early replicating fragile sites are mutated in cancer, my studies may help link R-loop- induced instability to tumorigenesis, which could provide new opportunities for cancer therapy. Overall, my proposed studies may greatly expand our knowledge in this emerging field and provide a foundation for other studies exploring the possible role of R-loops in tumorigenesis.